Attempts to understand genetic disorders of lipoprotein metabolism, such as familial hypercholesterolemia and type III hyperlipoproteinemia, have so far been made by looking for abnormalities of protein structure and function in individuals with clearly defined diseases. Now, advances in recombinant DNA technology are making it possible to search for a defective gene even when no specific protein abnormality has been identified. Using this technology, we have recently isolated and completely characterized the normal human apolipoprotein A-I (apo A-I) gene and we showed that it is physically linked with the apolipoprotein C-III (apo C-III) gene. In addition, we showed that these genes are convergently transcribed and that a DNA insertion in the apo A-I gene is related to apo A-I-apo C-III deficiency in the plasma of patients with premature atherosclerosis. The specific objectives in this proposal are (a) to isolate and characterize the apo A-I-apo C-III gene complex from the apo A-I-apo C-III deficient patients, (b) to isolate, characterize and study the origin of the inserted DNA in the apo A-I gene of these patients, and (c) to investigate the possible effect of this DNA insertion in the expression of apo A-I and apo C-III genes of these patients. This analysis will take place by constructing a genomic library from these patients from which both apo A-I and apo C-III genes can be isolated and sequenced. Functional analysis of the apo A-I-apo C-III complex from both normal individuals and these patients will be studied by using the papilloma virus eukaryotic vector system. The same genomic library constructed from the apo A-I-apo C-III deficient patients will be used to isolate and study the DNA insertion occurring in their apo A-I gene. Abnormalities in lipoprotein metabolism contribute significantly to coronary atherosclerosis and heart disease which are the number one cause of death in the United States. The type of studies proposed here have already produced a simple test by which diagnosis of homozygotes or heterozygotes for the DNA insertion in the apo A-I gene related to the development of premature atherosclerosis can be carried out. In addition, elucidation of the mechanisms by which these genes are controlled may provide the basis by which early diagnosis and treatment of atherosclerosis may be accomplished.